P433: PHARMACOLOGICAL DUAL INHIBITION OF PARP AND TIP60 AS A PROSPECTIVE THERAPEUTIC REGIMEN INDUCES SYNERGISTIC AML CELL DEATH
Authors: Tae Ju Park, Helen Wheadon, Laura Monaghan, Xu Huang, Heather Jorgensen
Journal: HemaSphere (2023)

Background: Synergistic leukemia cell death via simultaneous inhibition of lysine demethylase 4A (KDM4A) and poly ADP ribose polymerase (PARP) targeting dysregulation of DNA damage response (DDR) pathways was previously confirmed in our lab. It is hypothesized that co-targeting Tip60, a family member of histone acetyltransferase (HAT) that mediates DDR signals, with PARP inhibitors (PARPi), may introduce synergistic leukemic cell death in AML. Aims: Our project aims to characterize the molecular mechanisms of synergy between Tip60i and PARPi (‘TPARi’), and to elucidate their downstream targets as potential prognostic markers in AML. Methods: AML cell lines and patient cells were treated with TPARi to determine phenotypic changes using cellular assays. Molecular mechanisms were identified via pathway analysis of mRNA-seq data produced from dual drug-treated AML cells. Cross-validation between patient survival and gene expression of downstream target genes of TPAR using clinical datasets was used to show the importance of the downstream genes as prospective prognostic markers in AML patient survival. Results: Combinatorial loss of TPAR enzymatic activities induced synergistic AML cell death, validated by Bliss independence and Loewe additivity models (combination indices (CI)<1). TPARi induced phenotypic changes including increased apoptosis, cell cycle arrest, decreased colony formation, and accumulation of DNA damage markers, γH2A.X and cleaved PARP (p<0.0001). This was validated in AML patient cells where the combination treatment resulted in inhibition of proliferation. RNA-seq pathway analysis revealed that TPARi resulted in significantly enriched genes (p<0.05) involved in the downregulation of DDR sub-pathways, including mismatch repair (MMR; NES=-1.54; FDR=0.088) and interstrand crosslink repair (ICLR; NES=-1.25; FDR=0.22), in parallel. In addition, global pathway analysis delineated that the synergistic effect yielded upregulation of signaling pathways including, hypoxia, unfolded protein response, p53 pathway, and apoptosis; and downregulation of oxidative phosphorylation, glycolysis, and Myc pathway. Furthermore, Kaplan-Meier survival and Cox proportional hazards regression models showed a minimum subset of 13 genes as downstream target genes by TPAR (‘TPAR13’) successfully stratified in patient survival of clinical datasets (GSE12417, p<0.001; GSE37642, p<0.05; GSE6891, p<0.05; BeatAML, p<0.0001; TARGET_AML, p<0.05). Interestingly, when these datasets were divided by molecular characteristics, AML patients with myelodysplasia-related changes showed successful stratification of patient survival (p<0.005). Conclusion: Loss of cellular potential to repair DNA damage at the transcriptional level by interfering in MMR and ICLR pathways potentiates the gradual accumulation of DNA damage and effective AML cell death upon the combination treatment of TPARi. Transcriptional outcomes induced by TPARi lead to dysregulation of genes involved in oxidative phosphorylation for oxidation of nutrients and ATP production in glycolysis. These changes could potentially result in genomic instability, accumulation of global DNA lesion, and an increase in the cellular response to stress or unfolded protein. Overall, DNA damage accumulation with the attenuated cellular ability to repair damaged DNA, may increase apoptosis facilitated by downregulation of an oncogenic Myc pathway and upregulation of tumor suppressor p53 pathway. Importantly, this novel TPAR13 gene signature, as downstream targets of TPAR, has prognostic significance that successfully stratifies patient outcomes of AML. Keywords: Gene expression profile, Acute myeloid leukemia, Synergy, Survival prediction